Design, synthesis and biological evaluation of novel HIV-1 protease inhibitors with pentacyclic triterpenoids as P2-ligands

Article abstract of DOI:10.1016/j.bmcl.2018.12.040

The design, synthesis and SAR study of a new series of HIV-1 protease inhibitors with pentacyclic triterpenoids as P2 ligands and phenylsulfonamide as P2′ ligands were discussed. These compounds exhibited micromolar inhibitory potency, among which compound T1c displayed HIV-1 protease inhibition with IC₅₀ values of 0.12 μM, which was 67 times the inhibitory activity of its raw material Ursolic acid (8.0 μM).

Full text of DOI:10.1016/j.bmcl.2018.12.040

Accepted Manuscript
Design, synthesis and biological evaluation of novel HIV-1 protease inhibitors
with pentacyclic triterpenoids as P2-ligands
Mei Zhu, Xiao-Nan Du, Yun-Ge Li, Guo-Ning Zhang, Ju-Xian Wang, Yu-
Cheng Wang
PII:
S0960-894X(18)30987-9
https://doi.org/10.1016/j.bmcl.2018.12.040
BMCL 26209
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
3 September 2018
13 December 2018
17 December 2018
Please cite this article as: Zhu, M., Du, X-N., Li, Y-G., Zhang, G-N., Wang, J-X., Wang, Y-C., Design, synthesis
and biological evaluation of novel HIV-1 protease inhibitors with pentacyclic triterpenoids as P2-ligands,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.12.040
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Design, synthesis and biological evaluation of novel HIV-1 protease
inhibitors with pentacyclic triterpenoids as P2-ligands
Mei Zhu a, 1, Xiao-Nan Du a, 1, Yun-Ge Li b, Guo-Ning Zhang a, Ju-Xian Wang a,
Yu-Cheng Wang a, *
a
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and
Peking Union Medical College, Beijing 100050, China
b School of Pharmacy, Jinzhou Medical University, Jinzhou, Liaoning 121001, China
Abstract: The design, synthesis and SAR study of a new series of HIV-1 protease
inhibitors with pentacyclic triterpenoids as P2 ligands and phenylsulfonamide as P2'
ligands were discussed. These compounds exhibited micromolar inhibitory potency,
among which compound T1c displayed HIV-1 protease inhibition with IC50 values of
0.12 μM, which was 67 times the inhibitory activity of its raw material Ursolic acid
(8.0 μM).
Keywords: HIV-1 protease inhibitors; P2 ligand; pentacyclic triterpenoids
AIDS has been one of the most challenging problems in medicine since the first
1
case was reported in 1981 in USA. There were approximately 36.7 million people
living with AIDS worldwide by the end of 2017. Miserably, the morbidity and
mortality increased year by year. More than thirty kinds of drugs have been thus far
approved for the treatment of HIV/AIDS targeting different steps of HIV viral life
2
cycle. However, there still exists severe problems, such as the emergence of
3-5
extensively drug resistant and adverse effects.
Thus, there is an urgent need for
new anti-HIV drug candidates with increased potency, improved pharmacokinetic
properties, and reduced side effects.
Furthermore, as there exist lots of problems of traditional chemical drugs,
6
compounds of plant origin are brought into focus. Over 40% of antiviral drugs in
clinic were natural products or designed using natural products as prototypes from
1981 to 2014. 7 Pentacyclic triterpenoids are a large family of natural compounds with
varieties of biological activities such as antitumor, antiviral, antimicrobial,
anti-inflammatory and so on. 8,9 Lots of pentacyclic triterpenoids and their saponin or
modifying agents have been reported for their anti-HIV activity. 10-13
HIV-1 protease is one of the most important enzymes in the life cycle of HIV-1
virus, which would influence maturation of HIV-1 if it is inhibited. In this work, the
HIV-1 protease inhibitor Darunavir (DRV) was chosen as the lead compound and
* Corresponding author. Tel. /Fax: +86-10-63165263 (Y.-C. Wang).
E-mail address: wangyucheng@imb.pumc.edu.cn (Y.-C. Wang).
1 These authors contributed equally to this work.

pentacyclic triterpenoids were introduced into P2 ligands by scaffold-hopping strategy.
Meanwhile, phenylsulfonamide P2' ligands were investigated to maximize the
ligand-binding site interactions in the protease active site (Fig. 1). A series of novel
HIV-1 protease inhibitors were synthesized and biological evaluated.

Fig. 1. Chemical structure of target molecules
Compounds 9-13 were prepared from commercial available material (2S, 3S)-1,
2-epoxy-3-(boc-amino)-4-phenylbutane (8) as reported in the literatures in Scheme 1.
14-16 Treatment of epoxide 8 with isobutylamine followed by coupling with sulfonyl
chloride affording sulfonamides 10 and 11. Removal of the Boc protection of 10 and
11 was treated by trifluoroacetic acid to afford intermediate compounds 12 and 13.
Catalytic hydrogenation of 13 over 10% Pd/C in methanol afforded the corresponding
aminosulfonamide derivative 14.
Scheme 1. Syntheses of amines 12-14. Reagents and conditions: (a) i-BuNH2, CH3CN, 80 °C, 6 h;
(b) Aryl sulfonyl chloride, DIEA, DMAP(Cat.), THF, 0 °C ~ r.t, 3- 5 h; (c) CH2Cl2-CF3COOH
(1:1), 0 °C ~ r.t, 3 h; (d) H2 (gas), 50 psi, 10% Pd/C, CH3OH, r.t, 2 h.
The syntheses of inhibitors T1-T7 shown in Scheme 2 were carried out by
coupling acids 1-7 with amines 12-14 under an EDCI/HOBt/DMAP-mediated
coupling method. 14-16 The structures of these compounds are shown in Scheme 2.

Scheme 2. Syntheses of inhibitors T1-T7. Reagents and conditions: (e) EDCI, HOBt, DMAP,
anhydrous DMF, Ar, 0 °C ~ r.t, 12 h.
The inhibition potency of the synthetic compounds against HIV-1 protease was
17,18
evaluated using a fluorescence resonance energy transfer (FRET) method.
Inhibitors were further evaluated in cytotoxicity assay using a cell counting kit-8
assay. 19 The results were presented in Tables 1, 2 and 3, respectively.
Table 1 Enzymatic inhibitory activities and cytotoxicity of inhibitors with pentacyclic
triterpenoids amine-acetamide P2 ligands (1)
Compd.
T1a
R1
H
R2
R3
H
R4
R5
R6
H
Rb
IC50a (μM)
CC50
a(μM)
＞100
SI b
β-OH,
CH3
CH3
OCH3
0.46±0.12
＞217

α-H
β-OH,
α-H
T1b
T1c
T2a
T2b
T2c
T4a
T4b
T4c
H
H
H
H
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
NO2
NH2
0.40±0.15
0.12±0.04
0.24±0.09
0.15±0.08
0.17±0.09
0.15±0.09
0.17±0.11
0.30±0.19
＞100
＞100
＞100
＞100
86.85
＞100
＞100
＞100
＞250
＞833
＞417
＞667
511
β-OH,
α-H
H
β-OH,
α-H
H
CH3
CH3
CH3
H
OCH3
NO2
H
β-OH,
α-H
H
H
H
β-OH,
α-H
H
H
NH2
α-OH
α-OH
α-OH
β-OH,
α-H
OH
OH
OH
CH3
CH3
CH3
OCH3
NO2
＞667
＞588
＞333
β-OH,
α-H
H
β-OH,
α-H
H
NH2
T5a
T5b
T5c
H
H
H
-
O
H
H
H
-
CH3
CH3
CH3
-
CH3
CH3
CH3
-
H
H
H
-
OCH3
NO2
NH2
-
0.37±0.29
0.22±0.19
2.37±0.79
＞100
＞100
＞100
＞270
＞455
＞42.2
＞
O
O
DRV
-
0.57±0.17nM ＞100
175000
a Values were means of three independent experiments.
b SI = CC50/IC50
Table 2 Enzymatic inhibitory activities and cytotoxicity of inhibitors with pentacyclic
triterpenoids amine-acetamide P2 ligands (2)
Compd.
T3a
Rb
OCH3
NO2
NH2
-
IC50a (μM)
0.21±0.02
CC50 a(μM)
＞100
SI b
＞476
＞95
T3b
1.05±0.45
＞100
T3c
0.61±0.28
＞100
＞164
＞217000
DRV
0.46±0.12 nM
＞100
a Values were means of three independent experiments.
b SI = CC50/IC50

Table 3 Enzymatic inhibitory activities and cytotoxicity of inhibitors with pentacyclic
triterpenoids amine-acetamide P2 ligands (3)
Compd.
T6a
R7
α-H
α-H
α-H
β-H
β-H
β-H
-
Rb
OCH3
NO2
NH2
OCH3
NO2
NH2
-
IC50a (μM)
1.85±1.76
1.08±0.51
0.49±0.51
0.48±0.32
0.50±0.34
0.58±0.42
0.46±0.12 nM
CC50 a(μM)
＞100
74.90
SI b
＞54.1
69.4
T6b
T6c
＞100
＞100
＞100
＞100
＞100
＞204
＞208
＞200
＞172
＞217000
T7a
T7b
T7c
DRV
a Values were means of three independent experiments.
b SI = CC50/IC50
As shown above, the series of pentacyclic triterpenoids derivatives exhibited
unsatisfactory micromolar inhibitory potency. The reason might be that fragments of
pentacyclic triterpenoids were massive which were not matched with the residues of
20
the protease S2 subsite.
Furthermore, these fragments contained only a few
hydroxyl groups which could not promote extensive H-bonding interactions involved
directly or water-mediated with the backbone amino groups of residues Asp29 and
21
Asp30 of PR in the corresponding S2 subsite. However, there still existed some
regularity. For example, inhibitors with asiatic acid amine-acetamide P2 ligands with
more hydroxyl groups exhibited better inhibitory potency compared with ursonic acid
series, which might make more additional hydrogen bond interactions with the
backbone atoms and amino acid residues in the protease S2 subsite through oxygen
atoms. In addition, inhibitors with 18 β- glycyrrhetinic acid amine-acetamide P2
ligands along with 4-methoxylphenylsulfonamide and 4- nitrophenylsulfonamide as
P2' ligands exhibited better inhibitory potency compared with that of 18 α-
glycyrrhetinic acid, which indicated that the steric configuration of these compounds
might affect the combination between the inhibitors and the residues of the protease
S2 subsite, i. e. T7a vs T6a, T7b vs T6b.
In summary, a new series of HIV-1 protease inhibitors with pentacyclic
triterpenoids were designed and synthesized. Although compound T1c displayed 67
times the inhibitory activity of its raw material Ursolic acid (8.0 μM), these

compounds showed unsatisfactory micromolar inhibitory potency. But the results
gave a conclusion that massive structures in P2 ligands might be not matched with the
residues of the protease S2 subsite well, which was available for us to avoid
introducing massive structures in the following designing of HIV-1 protease inhibitors.
Furthermore, fragments of pentacyclic triterpenoids might be effective to other targets
of HIV, rather than HIV protease, which should be studied further.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Nos.
81473099, 81703366 and 81473098) and CAMS Innovation Fund for Medical
Sciences (CIFMS 2016-I2M-1-011, CIFMS 2016-I2M-3-014 and CIFMS
2017-I2M-3-019).
Supplementary data
Supplementary data associated with this article can be found in the online version.
The data include MOL files of the compounds described in this article.
References and notes
1. Friedman, K.A.; Laubenstein, L.; Marmor, M. MMWR Morb Mortal Wkly Rep. 1981, 30, 305.
2. Zhan, P.; Pannecouque, C.; Clercq, E. D.; Liu, X. J. Med. Chem. 2016, 59, 2849.
3. E. D. Clercq. Curr. Opin. Pharmacol 2010, 10, 507.
4. Clercq, E. D. Adv. Pharmacol 2013, 67, 317.
5. Clercq, E. D. Pharmacol 2013, 86, 711.
6. Xiao, S.; Tian, Z.; Wang, Y.; Si, L.; Zhang, L.; Zhou, D. Med. Res. Rev. 2018, 38, 951.
7. Newman, D. J.; Cragg, G. M. J Nat Prod. 2016, 79, 629.
8. Kvasnica, M.; Urban, M.; Dickinson, N. J.; Sarek, J. Nat. Prod. Rep. 2015, 32, 1303.
9. Li, Z.; Min, Q.; Huang, H.; Liu, R.; Zhu, R.; Zhu, Q. Bioorg Med Chem Lett. 2018, 28, 1501.
10. Hirabayashi, K.; Iwata, S.; Matsumoto, H.; Mori, T.; Shibata, S.; Baba, M.; Ito, M.; Shigeta, S.;
Nakashima, H.; Yamamoto N. Chem Pharm Bull. 1991, 39, 112.
11. Fields, A. P.; Bednarik, D. P.; Hess, A.; May, W. S. Nature 1988, 333, 278.
12. Sun, I.C.; Chen, C.H.; Kashiwada, Y.; Wu, J. H.; Wang, H. K.; Lee, K. H. J Med Chem. 2002,
45, 4271.
13. Bedoya, L. M.; Beltran, M.; Garcia-Perez, J.; Obregon-Calderon, P.; Callies, O.; Jimenez, I. A.;
Bazzocchi, I. L.; Alcami, J. Front in Pham. 2018, 9, 358.
14. Yang, Z. H.; Bai, X.G. ; Zhou, L.; Wang, J. X.; Liu, H. T.; Wang Y. C. Bioorg Med Chem Lett.
2015, 25, 1880.
15. Bai, X.; Yang, Z.; Zhu, M.; Dong, B.; Zhou, L.; Zhang, G.; Wang, J.; Wang, Y. Eur. J. Med.
Chem. 2017, 137, 30.
16. Ghosh, A. K.; Sridhar, P. R.; Leshchenko, S.; Hussain, A. K.; Li, J.; Kovalevsky, A. Y.;
Walters,D. E.; Wedekind, J. E.; Tokars, V. G.; Das, D.; Koh, Y.; Maeda, K.; Gatanaga, H.; Weber,
I. T.; Mitsuya, H. J. Med. Chem. 2006, 49, 5252.
17. Matayoshi, E. D.; Wang, G. T.; Krafft, G. A.; Erickson, J. Science 1990, 247, 954.

18. Gregson, S. J.; Howard, P. W.; Hartley, J. A.; Brooks, N. A.; Adams, L. J.; Jenkins, T. C.;
Kelland, L. R.; Thurston, D. E. J. Med. Chem. 2001, 44, 737.
19. Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto, T.; Suzuki, K.; Watanabe,
M. Anal. Commun. 1999, 36, 47.
20. Parai, M. K.; Huggins, D. J.; Cao, H.; Nalam, M. N.; Ali, A.; Schiffer, C. A.; Tidor, B.; Rana,
T. M. J. Med. Chem. 2012, 55, 6328.
21. Ghosh, A. K.; Parham, G. L.; Martyr, C. D.; Nyalapatla, P. R.; Osswald, H. L.; Agniswamy, J.;
Wang, Y. F.; Amano, M.; Weber, I. T.; Mitsuya, H. J. Med. Chem. 2013, 56, 6792.